Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Si-Miao-Yong-An decoction attenuates cardiac fibrosis via suppressing TGF-β1 pathway and interfering with MMP-TIMPs expression.

doi: 10.1016/j.biopha.2020.110132

Figure Lengend Snippet: Fig. 4. SMYAD improved cardiac fibrosis by inhibiting TGF-β1/Smad pathway (A) Representative immunoblots of protein in TGFβ1/Smad pathway. Western-blot analysis of (B) TGF-β1, (C) p-Smad2, (D) p-Smad3, (E) Smad4, (F) Smad7. Sham (n=6), TAC (n=6), TAC + Captopril (n=6), TAC + SMYAD (n=6). ### P < 0.001, ## P < 0.01, # P < 0.05 vs the Sham group; *** P < 0.001, ** P < 0.01, * P < 0.05 vs the TAC group.

Article Snippet: After blocking with 5 % non-fat milk for 1 h at room temperature, the blots were incubated overnight at 4 °C with the following antibodies: Col1 (Collagen Type I, 1:1000, Proteintech Cat 14695-1-AP); Col3 (Collagen Type III (N-Terminal), 1:1000, Proteintech Cat 22734-1-AP); αSMA (α-Smooth Muscle Actin, 1:1000, Cell Signaling Technology Cat 19245); MMP1 (MMP1, 1:1000, Proteintech Cat 10371-2-AP); MMP9 (MMP9 (N-Terminal), 1:1000, Proteintech Cat 10375-2-AP); TIMP1 (TIMP1, 1:1000, ab179580, Abcam); TIMP2 (TIMP2, 1:1000, ab180630, Abcam); CTGF (CTGF, 1:1000, Proteintech Cat 23936-1-AP); TGFβ1, (TGFβ1, 1:1000, Cell Signaling Technology Cat 3711); p-SMAD2 (Phospho-SMAD2, 1:500, Cell Signaling Technology Cat 18338); t-SMAD2 (SMAD2, 1:1000, Proteintech Cat 12570-1-AP); p-SMAD3 (Phospho-SMAD3, 1:500, Cell Signaling Technology Cat 9520); t-SMAD3 (SMAD3, 1:1000, Proteintech Cat 25494-1-AP); SMAD4 (SMAD4, 1:1000, Proteintech Cat 10231-1-AP); SMAD7 (SMAD7, 1:1000, Proteintech Cat 25840-1-AP); p-TAK1 (Phospho-TAK1, 1:1000, Cell Signaling Technology Cat 9339); t-TAK1 (TAK1, 1:1000, Cell Signaling Technology Cat 5206); p-p38 (Phospho-p38 MAPK, 1:1000, Cell Signaling Technology Cat 4511); t-p38 (P38 MAPK, 1:1000, Proteintech Cat 14064-1-AP); GAPDH (1:1,000, Cell Signaling Technology Cat 2118) was used as protein loading controls for shown in the figures.

Techniques: Western Blot

Journal: Kidney international

Article Title: Inhibition of transforming growth factor β1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice.

doi: 10.1016/j.kint.2021.02.035

Figure Lengend Snippet: Figure 4 | Lineage tracing for platelet-derived growth factor receptor b–positive (PDGFR-bD) cells was performed using PDGFR- bCreERT2/D mT/mG mice as controls without transforming growth factor b receptor 2 (TGFb-R2) deletion and in mice with tamoxifen- inducible deletion of TGFb-R2 in PDGFR-bD cells (TGFb-R2ko) with the mT/mG reporter. After tamoxifen treatment, all cells with Cre activation start to express green fluorescent protein (GFP). (Top row) Immunohistochemistry for a-smooth muscle actin (a-SMA) (red; left), GFP (green; middle), and merge (right) for lineage tracing of matrix-producing PDGFR-bþ cells on kidney tissue of a TGFb-R2ko mT/mG mouse after 5-day unilateral ureteral occlusion (UUO). Costaining of a-SMA showed a good overlap with GFP in a majority of myofibroblasts identified by a-SMA. These cells were targeted by the deletion of TGFb-R2. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (blue). Bars ¼ 50 mm. (Bottom row) Immunohistochemistry for the active nuclear transcription-factor phosphorylated SMAD3 (pSMAD3) (red) and GFP (green) on kidney sections of PDGFR-bCreERT2/þ mT/mG mice (left) and TGFb-R2ko mice (right) after 5-day UUO. In control kidneys, a robust nuclear pSMAD3 signal as an indicator for an activated TGFb pathway could be detected in tubules and GFPþ interstitial cells (arrows) after 5-day UUO. In TGFb-R2ko mice, GFPþ interstitial cells showed no activated phosphorylated SMAD3 (pSMAD3) signal (arrows), whereas the tubular pSMAD3 signal was preserved. Bars ¼ 20 mm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Article Snippet: Chicken anti– green fluorescent protein (ab13970, Abcam) and rabbit anti– phosphorylated SMAD3 (#9520S, Cell Signaling Technology) immunohistochemistry was performed analogously.

Techniques: Derivative Assay, Activation Assay, Immunohistochemistry, Control

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: A novel ocular function for decorin in the aqueous humor outflow.

doi: 10.1016/j.matbio.2021.02.002

Figure Lengend Snippet: Fig. 7. Decorin inhibits activation of the SMAD-signaling pathway by TGF-b2. A) Treatment with 4 pMTGF-b2 or 3 nM CTGF led to increased phosphorylation of SMAD2 in HTMN cells after 4 h. Pre-incubation with 25 nM DCN blocked the activating effect of TGF-b2 almost completely and reduced the activating effect of CTGF. B) Chemiluminescence signal for pSMAD2 was increased after treatments with TGF-b2 and CTGF, while the signal for SMAD2 was unchanged. The increased phosphorylation of SMAD2 could be circumvented by pre-incubation with DCN. C) Quantification of pSMAD2 and SMAD2 signals showed a significant increase in the pSMAD2/SMAD2 ration after treatment with TGF-b2 (1.48 § 0.46, n = 5, p = 0.047), but not after treatment with CTGF (1.41 § 0.45, n = 5, p = 0.07). Treatment with 25 nM DCN did not affect the pSMAD2/SMAD2 ratio (1.12 § 0.23, n = 5, p = 0.26). Pre-treatment with 25 nM DCN circumvented the activating effect of TGF-b2 on the SMAD-Signaling pathway (pSMAD2/SMAD2 = 1.16 § 0.55, n = 5, p = 0.53). (D) pSMAD2 staining intensity was markedly stronger in the anterior eye of 12-week old Dcn/- mice compared to wildtype lit- termates. pSMAD2 signal was increased in the outflow tissues but also in the ciliary muscle and the cornea. (Co = Cornea, TM = Trabecular meshwork, CM = Ciliary muscle. Red = pSMAD2, blue = DAPI, scale bars: 20 mm).

Article Snippet: Antibodies were used as follows: rabbit anti-human/ mouse-DCNH80 (1:200), goat anti-CCN2/CTGF (1:500), rabbit anti-FNH300 (1:500), donkey antirabbit-horseradish peroxidase (HRP), chicken antirabbit-AP and chicken anti-goat-HRP (1:2000; all Santa Cruz, CA, USA), chicken anti GFAP (1:10,000, LS Bio, Seattle, USA), rabbit anti pSMAD2 (1:1000, Cell Signaling, Denvers, USA), rabbit anti SMAD2 (1:1000, Cell Signaling, Denvers, USW).

Techniques: Activation Assay, Phospho-proteomics, Incubation, Staining

Journal: Life science alliance

Article Title: Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

doi: 10.26508/lsa.201900581

Figure Lengend Snippet: Figure 2. cDCs and cDC progenitors: CD115+ CDP (Lin2CD117int), SiglecH2Ly6C+, and SiglecH2Ly6C2 pre-DCs are increased in Smad32/2 mice. Immunophenotyping of Smad3−/−(black circles) or Smad3+/+ (white circles) mice (n = 6/genotype) was performed using flow cytometry. (A) Representative contour plots show CD11c+MHCII+, CD11b+CD11c+MHCII+, and CD8+CD11c+MHCII+ cells in BM, spleens (SP), superficial lymph nodes, and mesenteric lymph nodes. Dots in the graphs show the numbers of CD11c+MHCII+, CD11b+CD11c+MHCII+, and CD8+CD11c+MHCII+ cells in BM, SP, superficial lymph nodes, and mesenteric lymph nodes of each mouse. Horizontal bars show means. (B) Representative contour plots show the expression of CD115/CD135 in Lin−Sca-1−CD117hi or Lin−Sca-1−CD117int gates. Dots in the graphs show the numbers of Lin−Sca-1−CD117hiCD115+CD135+ MDPs and Lin−Sca-1−CD117intCD115+CD135+ CDPs in the BM of each mouse. (C) Bar graphs show the expression levels of Cx3cr1 and Dngr1 mRNA in Lin−CD115+ BM cells detected using RT–qPCR with means + s.d. Black bars represent Smad3−/−, whereas white bars represent Smad3+/+ mice. Representative contour plots show CX3CR1+CD370+Lin−CD117intCD115+CD135+ BM cells. (D) Representative contour plots show the expression of SiglecH/Ly6C in the CD11c+MHCII−CD135+CD172α−pre-DC gate. Dots in the graphs show the numbers of CD11c+MHCII−CD135+CD172α−SiglecH−cells in BM. P-values were calculated by a two-tailed unpaired t test.

Article Snippet: For the PLA, fixed cells were permeabilized by 0.1% Triton X-100 in PBS, and stained with rabbit antibodies against SMAD2, SMAD3, phospho-SMAD2 (S465/467), phospho-SMAD3 (S423/425) (Cell Signaling Technology), STAT3 (clone C-20; Santa Cruz Biotechnology), c-SKI (clone 6D763; Santa Cruz Biotechnology), and FLAG (clone 8H8L17; Invitrogen).

Techniques: Cytometry, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Life science alliance

Article Title: Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

doi: 10.26508/lsa.201900581

Figure Lengend Snippet: Figure 7. Interaction of c-SKI with R-SMADs and phosphorylated STAT3 is essential for repression of SMAD3 and cDC differentiation. (A) Effects of c-SKI mutations (Δ2/3 that does not interact with Smad2/3 and W274E that does not interact with Smad4) on the Smad3 promoter activity in CD11b+ FLT3L- induced or CD11b+ GM-CSF plus IL-4–induced BMDCs transfected with the indicated plasmids were determined by the Smad3 promoter luciferase reporter assay. (B) Effects of STAT3 phosphorylation site-specific mutants (Y705F and S727A) on SMAD2/3-induced activation of the Smad3 promoter constructs transfected with the indicated plasmids in CD11b+ FLT3L-induced or CD11b+ GM-CSF plus IL-4–induced BMDCs were determined by the Smad3 promoter luciferase reporter assay. (C) Contour plots show the expression of CD11c/MHCII and CD11b/CD24 in the CD11c+MHCII+ gate of FLT3L-induced or GM-CSF plus IL-4–induced BMDCs transfected with the indicated c-SKI mutants, c-SKI siRNA and control pcDNA or control siRNA. (D) Contour plots show the expression of CD11c/MHCII and CD11b/CD24 in the CD11c+MHCII+ gate of FLT3L- induced or GM-CSF plus IL-4–induced BMDCs transfected with the indicated STAT3 phosphorylation site-specific mutants (Y705F and S727A) and STAT3 siRNA or control siRNA 4 h before culture and analysed on day 8. Luciferase reporter assays were performed in triplicate. Data are representative of three independent experiments. Graphs show means + s.d. P-values were calculated by a two-tailed unpaired t test. ***P < 0.0005.

Article Snippet: For the PLA, fixed cells were permeabilized by 0.1% Triton X-100 in PBS, and stained with rabbit antibodies against SMAD2, SMAD3, phospho-SMAD2 (S465/467), phospho-SMAD3 (S423/425) (Cell Signaling Technology), STAT3 (clone C-20; Santa Cruz Biotechnology), c-SKI (clone 6D763; Santa Cruz Biotechnology), and FLAG (clone 8H8L17; Invitrogen).

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Phospho-proteomics, Activation Assay, Construct, Expressing, Control, Two Tailed Test